Enzymatic process for the manufacture of l-ascorbic acid and d-erythorbic acid

ABSTRACT

A process for producing L-ascorbic acid from  2 -keto-L-gulonic acid or D-erythorbic acid from 2-keto-D-gluconic acid by contacting 2-keto-L-gulonic acid or 2-keto-D-gluconic acid, respectively, with an enzym having α-amylase activity in solution. The solvent for this reaction can be water, an aqueous alcohol, an organic solvent or a mixture thereof. In each case, the starting material can be in the form of the free acid, the sodium salt, or the calcium salt.

The present invention relates to a novel process for the manufacture ofL-ascorbic acid or D-erythorbic acid from 2-keto-L-gulonic acid or2-keto-D-gluconic acid, respectively, by using an α-amylase or an enzymehaving α-amylase activity.

L-Ascorbic acid, also known as vitamin C, is widely used not only asmedicine but also in the field of food industry, cosmetics industry andthe like, and is a very useful compound. D-erythorbic acid is mainlyused as an antioxidant for food additives.

So far, L-ascorbic acid has been commercially produced by the well-knownReichstein method, in which L-ascorbic acid is produced from D-glucosevia D-sorbitol, L-sorbose, diacetone-L-sorbose,diacetone-2-keto-L-gulonic acid, 2-keto-L-gulonic acid, and methyl2-keto-L-gulonate. In this process, the conversion of D-sorbitol toL-sorbose is the sole microbial step, the others being chemical steps.The conversion of diacetone-2-keto-L-gulonic acid to L-ascorbic acid isachieved by two different procedures: (i) deprotection to give2-keto-L-gulonic acid, followed by esterification with methanol andbase-catalyzed cyclization; and (ii) acid-catalyzed cyclization toL-ascorbic acid directly from the protected or deprotected2-keto-L-gulonic acid. These conversion processes must be performed bynon- or low-water reaction systems. Environmentally and economically, areaction without organic solvents is preferred.

On the other hand, D-erythorbic acid has been produced from D-glucosevia 2-keto-D-gluconic acid, which itself can be produced by fermentationwith a strain belonging to the genus Pseudomonas, and via methyl2-keto-D-gluconate.

Much time and effort has been devoted to finding other methods ofproducing L-ascorbic acid by microorganisms. Most studies on microbialproductions of L-ascorbic acid have focused on the production of anintermediate of L-ascorbic acid production, 2-keto-L-gulonic acid, fromL-sorbose (e.g. EP 213,591; U.S. Pat. No. 4,960,695; EP 221,707), fromD-sorbitol (e.g. EP 213,591; U.S. Pat. No. 5,312,741; WO 95/23220; WO98/17819], or from D-glucose via 2,5-diketogluconic acid with a single,mixed or recombinant culture. The. 2-keto-L-gulonic acid can then beconverted into L-ascorbic acid by chemical means as described above.

As mentioned above, the chemical conversion of 2-keto-L-gulonic acid toL-ascorbic acid via 2-keto-L-gulonic acid γ-lactone is an acid-catalyzedreaction accompanying removal of a water molecule. The main principle ofthe reaction is a carboxyl ester bond formation to make the γ-lactonering in the 2-keto-L-gulonic acid molecule. Therefore, especially inwater phase, the final state in the equilibrium reaction is determinedby the physico-chemical conditions. Production of L-ascorbic acid from2-keto-L-gulonic acid by chemical conversion is considerable even in thewater phase, but is not sufficient for commercial application.Production process in water phase or in water phase with low organicsolvent content is highly desirable for cost effectiveness andenvironmental demand. Then, some biological enhancement on the chemicalconversion must bedesired for the production in water phase. Both hightemperature and acidic (low) pH are obviously preferable for thereaction.

WO 97/43433 describes a process for allegedly preparing L-ascorbic acidby contacting 2-keto-L-gulonic acid or an ester thereof with a hydrolaseenzyme catalyst selected from the group consisting of proteases (enzymeclass EC 3.4.x.x), esterases (enzyme class EC 3.1.x.x), lipases (enzymeclass EC 3.1.x.x) and amidases (enzyme class EC 3.5.x.x). Using ahydrolase such as a protease, an esterase, a lipase or an amidase, U.S.Pat. No. 6,022,719 exemplifies the formation of L-ascorbic acid from anester of 2-keto-L-gulonic acid such as butyl 2-keto-L-gulonate, but noapparent formation of L-ascorbic acid from 2-keto-L-gulonic acid itself.It does not disclose an α-amylase or an enzyme having α-amylase activityas the hydrolase enzyme catalyst for the purpose of L-ascorbic acidformation from 2-keto-L-gulonic acid.

α-Amylase is an endo-type hydrolase which catalyzes hydrolysis of theα-1,4-glucosidic linkages of starch and liberates poly- andoligosaccharide chains of varying lengths. α-Amylases are available froma wide variety of sources, microorganisms, plants, and animals. It hasbeen known that some α-amylases have good thermo-stability. Moreover,the thermoacidophilic bacterium Alicyclobacillus acidocaldarius producesan α-amylase, which is highly thermo-stable under acidic condition.

The present invention provides an enzymatic process for the productionof L-ascorbic acid from 2-keto-L-gulonic acid, which comprisescontacting 2-keto-L-gulonic acid in solution with an α-amylase.Moreover, the present invention also provides an enzymatic process forthe production of D-erythorbic acid from 2-keto-D-gluconic acid, whichcomprises contacting 2-keto-D-gluconic acid in solution with anα-amylase.

As used herein, the term “α-amylase” includes the α-amylase enzymeitself and enzymes having α-amylase activity.

The microorganisms “Bacillus amyloliquefaciens ”, “Bacilluslicheniformis”, and “Alicyclobacillus acidocaldarius” also includesynonyms or basonyms of such species having the same physico-chemicalproperties, as defined by the International Code of Nomenclature ofProkaryotes.

The α-amylases used as catalysts in the processes of the presentinvention are those obtained from organisms including animals, plants,and microorganisms such as fungi, yeast, and bacteria. The preferredα-amylases of the present invention are those of B. amyloliquefaciens,B. licheniformis, A. acidocaldarius, and porcine pancreas. Morepreferred is an enzyme having α-amylase activity of A. acidocaldarius,especially A. acidocaldarius ATCC 27009.

The α-amylases used in the processes of the present invention may bepurchased from suppliers such as Sigma-Aldrich Co. (St. Louis, USA) ormay be isolated from any appropriate organisms including animals,plants, and microorganisms by usual protein purification methods such asammonium sulfate precipitation, chromatography, and crystallization. Inthe processes of the present invention, any form of α-amylase can beused, in particular an enzyme solution or the immobilized enzyme.

In the processes of the present invention, the preferred chemical formof 2-keto-L-gulonic acid and 2-keto-D-gluconic acid are the free acidsor their metal salts such as the sodium and calcium salts.

The reaction mixture may contain a suitable stabilizer for (X-amylasesuch as a Ca ion and may further contain an antioxidant such as2-mercaptoethanol, dithiothreitol, or cysteine to prevent thedegradation of the produced L-ascorbic acid or D-erythorbic acid.

The reaction temperatures for the process of the present invention arein the range of from 0° C. to 120° C. The preferred temperatures are inthe range of from 20° C. to 100° C., and most preferably in the range offrom 37° C. to 90° C.

Suitable pHs for the processes of the present invention are in the rangeof from 1.5 to 12. The preferred pHs are in the range of from 1.5 to 8,and most preferably in the range of from 2 to 7. The pH of the reactionmixture may be adjusted by using suitable buffers or may be directlyadjusted by addition of acid e.g., HCl or alkali e.g., NaOH.

The reaction period is suitably in the range of from 1 to 7 days,preferably in the range of from 1 to 3 days.

The process of the present invention may be performed in a solvent suchas water, an aqueous alcohol, an organic solvent or a mixture thereof.Examples of an alcohol include a C₁-C₆ alcohol, such as methanol,ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, ortert.-butanol. Examples for organic solvents include aliphatichydrocarbons (e.g., heptane and iso-octane), alicyclic hydrocarbon(e.g., cyclohexane), and aromatic hydrocarbons (e.g., benzene andtoluene). The preferred solvent is water or an aqueous solvent.

The L-ascorbic acid or D-erythorbic acid produced under the conditionsas described above can easily be recovered by methods known in the art.For example, the L-ascorbic acid or D-erythorbic acid can be isolated bycrystallization, electrodialysis, or column chromatography by using ionexchange resin.

The present invention is explained in more detail by referring to thefollowing Examples; however, it should be understood that the presentinvention is not limited to those particular Examples.

EXAMPLE 1 Conversion of 2-keto-L-gulonic acid to L-ascorbic acid byα-amylases of B. amyloliquefaciens, B. licheniformis, and PorcinePancreas

The α-amylases of B. amyloliquefaciens (Sigma-Aldrich, product number10068), B. licheniformis (Sigma-Aldrich, product number A4551), andporcine pancreas (Sigma-Aldrich, product number A4268) were tested forthe conversion of 2-keto-L-gulonic acid to L-ascorbic acid. The enzymepowders were dissolved in buffer 1 [20 mM Na-MES buffer (pH 7.0) and 2mM CaCl₂] and used for the conversion reaction. The reaction mixturecontained 12% sodium 2-keto-L-gulonic acid monohydrate, 2 mM CaCl₂, andthe α-amylase in 50 μl of 100 mM buffer. The concentration of theα-amylase and the buffer in each reaction mixture is shown in Table 1.The reaction was carried out under anaerobic condition at 70° C. for 20hours.

L-Ascorbic acid was assayed by HPLC on YMC-Pack PolyamineII column (ID.4.6×150 mm; YMC Co., Japan) at 264 nm with the mobile-phase solventcontaining 70% (v/v) acetonitrile and 15 mM ammonium dihydrogenphosphateat a flow rate of 1.5 ml/min. Amounts of biologically producedL-ascorbic acid were determined by the difference between results fromthe reactions with and without the α-amylase. The amounts of L-ascorbicacid produced are summarized in Table 1. TABLE 1 L-Ascorbic acidα-Amylase* Enzyme (μg/ml) Buffer (pH) (mg/l) 10068 400 Na-MES (pH 6.0)29.1 A4551 320 Na-acetate (pH 5.0) 84.9 A4268 400 Na-MES (pH 5.5) 61.9*product number

EXAMPLE 2 Purification of an Enzyme Having α-amylase Activity from A.acidocaldarius

A. acidocaldarius ATCC 27009 was aerobically grown in 24.61 of a mediumconsisting of 9.8 mM (NH₄)₂SO₄, 0.47 mM CaCl₂, 2.7 mM KH₂PO₄, 1.0 mMMgSO₄, 10 μM FeSO₄, 9.1 μM MnCl₂, 11.7 μM Na₂B₄O₇, 0.57 μM ZnSO₄, 0.2 μMCuCl₂, 0.12 μM VOSO₄, 0.16 μM NaMoO₄, 30 nM CoSO₄, and 10 mM maltose (pH3.5) at 55° C. for 15 hours. The culture fluid was recovered bycentrifugation.

During the enzyme purification, all runs of column chromatography werecarried out at 4° C. The α-amylase activity was determined by thespectrophotometric assay by using p-nitrophenyl α-D-maltopentoside(Sigma-Aldrich) as a substrate. The assay mixture contained 1 mMp-nitrophenyl α-D-maltopentoside and an enzyme sample in 40 μl of 50 mMNa-acetate buffer (pH 4.0). After incubating at 55° C. for 20 minutes,100 μL of 1 M Tris-HCl buffer (pH 7.5) was added to the reactionmixture. The released p-nitrophenol was detected as an absorbance at 405nm.

The recovered culture fluid was subjected to DEAE Sepharose Fast Flow(Amersham Pharmacia Biotech UK Ltd. Buckinghamshire, UK) columnchromatography. 0.5 M Na-MES buffer (pH 6.0) was added to the culturefluid at 20 mM and the pH was adjusted to 6.0 with NaOH. The preparedfluid sample from 1.65 1 culture was loaded on DEAE Sepharose Fast Flowcolumn (100 ml: I.D. 4.4×6.6 cm) equilibrated with buffer 2 [20 mMNa-MES buffer (pH 6.0), 0.5 mM CaCl₂, and 1.0 mM MgSO₄]. After washingwith buffer 2, the enzyme was eluted with a linear gradient of NaCl(0-0.45 M in buffer 2). This step was repeated four times to treat 6.6 1of culture. The active fractions of four chromatography runs werebrought together and dialyzed against buffer 3 [20 mM Na-acetate buffer(pH 3.0), 0.5 mM CaCl₂, and 1.0 mM MgSO₄]. The dialyzed sample wasloaded on SP Sepharose Fast Flow column (80 ml: I.D. 4.4×5.3 cm,Amersham Pharmacia Biotech UK) equilibrated with buffer 3. After washingwith buffer 3, the enzyme was eluted with a linear gradient of NaCl(0-0.6 M in buffer 3). The active fractions were collected. Afterdialysis against buffer 2, the enzyme sample was loaded on Q SepharoseFast Flow column (30 ml: I.D. 2.5×6.2 cm, Amersham Pharmacia Biotech UK)equilibrated with buffer 2. After washing with buffer 2, the enzyme waseluted with a linear gradient of NaCl (0-0.8 M in buffer 2). The activefractions were collected and concentrated to 3.5 ml with CentriplusYM-30 (Millipore Co., USA). The concentrated sample was passed throughHiPrep Seph-acryl S-300 HR 16/60 (Amersham Pharmacia Biotech UK) withbuffer 4 [20 mM Na-MES buffer (pH 6.0), 0.5 mM CaCl₂, 1.0 mM MgSO₄, and0.15 M NaCl]. A fraction of 9 ml containing the enzyme having α-amylaseactivity was obtained. The enzyme sample was dialyzed against buffer 5[20 mM Na-MES buffer (pH 6.0), 0.5 mM CaCl₂, 1.0 mM MgSO₄, and 50 mMNaCl] and concentrated to 0.11 ml by repeated concentration/dilution ina concentrator with Centricon YM-30 (Millipore Co., USA). On SDS-PAGE,the purified enzyme mainly exhibited a molecular mass of approximately160 kDa. Finally, 0.47 mg of the enzyme having α-amylase activity withthe purity of about 80% was obtained.

EXAMPLE 3 Conversion of 2-keto-L-gulonic Acid to L-ascorbic Acid by anEnzyme Having α-amylase Activity of A. acidocaldarius

The enzyme having α-amylase activity purified from A. acidocaldarius inExample 2 was tested for the conversion of 2-keto-L-gulonic acid toL-ascorbic acid. The reaction mixture contained 10% sodium2-keto-L-gulonic acid monohydrate (adjusted to pH 2.5 with HCl) and theenzyme (100 or 200 μg/ml) in 20 μl. The reaction was carried out underanaerobic conditions at 80° C. for 24 hours. L-ascorbic acid was assayedby the method described in Example 1. Amounts of biologically producedL-ascorbic acid were determined by the difference between results fromthe reactions with and without the enzyme. 100 μg/ml enzyme produced3.90 g/l L-ascorbic acid, 200 μg/ml enzyme produced 4.22 g/l L-ascorbicacid.

EXAMPLE 4 Conversion of 2-keto-D-gluconic Acid to D-erythorbic Acid byα-amylases of B. amyloliquefaciens, B. licheniformis, and PorcinePancreas

The α-amylases of B. amyloliquefaciens, B. licheniformis, and porcinepancreas described in Example 1 were tested for the conversion of2-keto-D-gluconic acid to D-erythorbic acid. The α-amylases dissolved inbuffer 1 were used for the conversion reaction. The reaction mixturecontained 5% 2-keto-D-gluconic acid hemicalcium salt (Sigma-Aldrich), 4mM CaCl₂, and 400 μg/ml of the α-amylase per ml in 50 μl of 100 mMbuffer. The buffer in each reaction mixture is shown in Table 3. Thereaction was carried out under anaerobic conditions at 70° C. for 20hours. D-Erythorbic acid was assayed by the same method as forL-ascorbic acid (described in Example 1). Amounts of biologicallyproduced D-erythorbic acid were determined by the difference betweenresults from the reactions with and without the α-amylase. The amountsof D-erythorbic acid produced are summarized in Table 2. TABLE 2D-Erythorbic acid α-Amylase* Enzyme (μg/ml) Buffer (pH) (mg/l) 10068 400Na-acetate (pH 5.0) 64.7 A4551 400 Na-acetate (pH 5.0) 69.5 A4268 400Na-MES (pH 5.5) 60.5*product number

EXAMPLE 5 Conversion of 2-keto-D-gluconic Acid to D-erythorbic Acid byEnzymes Having α-amylase Activity of A. acidocaldarius

The enzyme purified from A. acidocaldarius in Example 2 was tested forthe conversion of 2-keto-D-gluconic acid to D-erythorbic acid. Thereaction mixture contained 5% 2-keto-D-gluconic acid hemicalcium salt(adjusted to pH 2.5 with HCl) and the enzyme (0 or 200 μg/ml) in 20 μl.The reaction was carried out under anaerobic conditions at 70° C. for 21hours. D-Erythorbic acid was assayed by the method described in Example4. The amount of biologically produced D-erythorbic acid was determinedby the difference between results from the reactions with and withoutthe enzyme. 76.0 mg/l of D-erythorbic acid was produced by the reactionwith the enzyme.

1. A process for preparing L-ascorbic acid from 2-keto-L-gulonic acid orpreparing D-erythorbic acid from 2-keto-D-gluconic acid which comprisescontacting a solution containing 2-keto-L-gulonic acid or2-keto-D-gluconic acid, respectively, with an α-amylase or an enzymehaving α-amylase activity.
 2. The process according to claim 1 whereinthe solvent is an aqueous solvent selected from the group consisting ofwater, an alcohol, and a mixture thereof.
 3. The process according toclaim 1 wherein 2-keto-L-gulonic acid or 2-keto-D-gluconic acid iscontacted with the enzyme at a temperature in the range of from 0° C. to120° C.
 4. The process according to claim 3 wherein 2-keto-L-gulonicacid or 2-keto-D-gluconic acid is contacted with the enzyme at atemperature in the range of from 20° C. to 100° C.
 5. The processaccording to claim 4 wherein 2-keto-L-gulonic acid or 2-keto-D-gluconicacid is contacted with the enzyme at a temperature in the range of from37° C. to 90° C.
 6. The process according to claim 1 wherein2-keto-L-gulonic acid or 2-keto-D-gluconic acid is contacted with theenzyme at a pH in the range of from 1.5 to
 12. 7. The process accordingto claim 6 wherein 2-keto-L-gulonic acid or 2-keto-D-gluconic acid iscontacted with the enzyme at a pH in the range of from 1.5 to
 8. 8. Theprocess according to claim 6 wherein 2-keto-L-gulonic acid or2-keto-D-gluconic acid is contacted with the enzyme at a pH in the rangeof from 2 to
 7. 9. The process according to claim 1 wherein2-keto-L-gulonic acid or 2-keto-D-gluconic acid is selected from thegroup consisting of the free acid and a metal salt.
 10. The processaccording to claim 1 wherein 2-keto-L-gulonic acid or 2-keto-D-gluconicacid is contacted with an enzyme having α-amylase activity at atemperature in the range of from 37° C. to 90° C. and at a pH in therange of from 2 to 7.